摘要
目的:建立稳定的CD34+脐血造血干细胞(CD34+UHSC)的体外分离、培养扩增体系,并研究其生物学特性。方法:密度梯度离心及免疫磁珠法从脐血中分离CD34+UH-SC,加入细胞因子SCF、IL-3和IL-6进行体外培养扩增,观察细胞形态及其生长特性。应用流式细胞仪测定细胞周期及其CD34分子的表达,研究其增殖、生长和分化特性。以RT-PCR法检测干细胞高表达基因ABCG2的转录表达。结果:在体外培养条件下,CD34+UHSC悬浮生长,约1周后易形成克隆球,增殖旺盛,呈现指数生长期;细胞增殖周期随体外培养时间延长而逐渐趋缓;CD34+UHSC表面标志亦随体外培养时间延长而表达下降,同时ABCG2基因的表达亦逐步下调。结论:体外获得纯化的CD34+UHSC,生长稳定、增殖能力强,但随体外培养时间的延长,CD34+分子标志逐步丢失,提示在增殖旺盛的指数生长期7～21d内最适用于各项相关实验研究。
Objective：To establish a stable culture system in vitro for isolating CD34＋ hematopoietic stem cells derived from human umbilical cord blood （CD34＋UHSCs） and to investigate their biological characteristics.Methods：Density-gradient centrifugation and immunomagnetic beads separation methods were used to derive CD34＋UHSCs from human umbilical cord blood,cultured with the cytokines of SCF,IL-3 and IL-6 and amplification in vitro to observe the cell morphology and biological properties.Flow cytometry was used to measure the cell cycle and detect the expression of CD34 for exploring the cell proliferation,growth and differentiation.RT-PCR was used to detect the transcription expression of highly expressed gene ABCG2.Results：In vitro,CD34＋UHSCs were presented in suspension growth and formed clone sphere in about 1 week and characterized by exponential phase of growth with strong proliferation.The cell division cycles became declined with prolonged length of culture in vitro,and the cell surface markers of CD34 hematopoietic stem cells died away with time extension and the ABCG2 expression became down-regulated simultaneously.Conclusion：The purified CD34＋UHSCs in vitro can grow stably and proliferate intensively,and yet,the cellular marker of CD34＋ became lost gradually with culture extension in vitro.The results suggest that CD34＋UHSCs in vigorous proliferation are feasible to be used in the correlated experimental study in the exponential phase of 7-21 days.
出处
《皖南医学院学报》
CAS
2010年第3期161-165,共5页
Journal of Wannan Medical College
基金
安徽省高校省级自然科学研究项目(KJ2010B238)
关键词
脐血
CD34^+造血干细胞
体外培养
流式细胞术
umbilical cord blood
CD34＋ hematopoietic stem cells
culture in vitro
flow cytometry
